WO1994018156A1 - Agents tensioactifs amines quarternaires et procedes d'utilisation de ceux-ci - Google Patents

Agents tensioactifs amines quarternaires et procedes d'utilisation de ceux-ci Download PDF

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WO1994018156A1
WO1994018156A1 PCT/US1994/000680 US9400680W WO9418156A1 WO 1994018156 A1 WO1994018156 A1 WO 1994018156A1 US 9400680 W US9400680 W US 9400680W WO 9418156 A1 WO9418156 A1 WO 9418156A1
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rna
surfactant
carbons
quaternary amine
group
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PCT/US1994/000680
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English (en)
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Donald E. Macfarlane
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University Of Iowa Research Foundation
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Priority claimed from US08/013,419 external-priority patent/US5300635A/en
Application filed by University Of Iowa Research Foundation filed Critical University Of Iowa Research Foundation
Priority to JP51806594A priority Critical patent/JP3615545B2/ja
Priority to AU62305/94A priority patent/AU6230594A/en
Publication of WO1994018156A1 publication Critical patent/WO1994018156A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/89—Carboxylic acid amides having nitrogen atoms of carboxamide groups quaternised
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C211/00—Compounds containing amino groups bound to a carbon skeleton
    • C07C211/62—Quaternary ammonium compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C211/00—Compounds containing amino groups bound to a carbon skeleton
    • C07C211/62—Quaternary ammonium compounds
    • C07C211/63—Quaternary ammonium compounds having quaternised nitrogen atoms bound to acyclic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C211/00—Compounds containing amino groups bound to a carbon skeleton
    • C07C211/62—Quaternary ammonium compounds
    • C07C211/64—Quaternary ammonium compounds having quaternised nitrogen atoms bound to carbon atoms of six-membered aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor

Definitions

  • This invention relates generally to the isolation of ribonucleic acids from blood and other biological samples, and more specifically to a method of isolation employing novel quaternary amine surfactants.
  • RNA ribonucleic acid
  • RNA isolating RNA include a variety of techniques to disrupt the cell and liberate RNA into solution and to protect RNA from RNases.
  • RNA is separated from the DNA and protein which is solubilized along with the RNA.
  • the use of the powerfully chaotropic salts of guanidinium to simultaneously lyse ⁇ cells, solubilize RNA and inhibit RNases was described in Chirgwin et al, Biochem..
  • RNA isolation methods free solubilized RNA of contaminating protein and DNA by extraction with phenol at an acidic pH using chloroform to effect a phase separation [D. M. Wallace, Meth. Enzym. , 152:33-41 (1987) ] .
  • a commonly used single step isolation of RNA involves homogenizing cells in 4M guanidinium isothiocyanate, followed by the sequential addition of sodium acetate, pH4, phenol, and chloroform/ isoamyl alcohol. After centrifugation, RNA is precipitated from the upper layer by the addition of alcohol [P. Chomczynski and N. Sacchi, Anal. Biochem..
  • RNA in white blood cells are likely to separate these cells from blood by centrifugal methods (typically through a gradient of Ficoll/hypaque) , and then apply one of the above described methods to the isolated cells. Thus, there is no established method for isolating RNA from whole blood. Similarly, investigators wishing to study viruses may separate viral RNA from plasma using such methods.
  • RNA in clinical practice is hampered by the difficulty of separating RNA from the protein and DNA in the cell before the RNA is degraded by nucleases, such as RNase.
  • RNase and other nucleases are present in blood in sufficient quantities to destroy unprotected RNA in a few seconds.
  • Successful methods for the isolation of RNA from cells must be capable of preventing hydrolysis of RNA by nucleases.
  • the present invention provides a novel method for isolating RNA from a biological sample, including blood, involving the use of an aqueous, cationic surfactant solution comprising a selected quaternary amine.
  • the selected quaternary amine is produced through the reaction of a quaternary amine hydroxide and an acid of the group consisting of phosphoric, sulfuric, formic, acetic, propionic, oxalic, malonic, succinic and citric.
  • the quaternary amine is either an acyltrimethylammonium or an acylbenzyldimethylammonium, where the acyl group contains 12, 14, 16 or 18 carbons.
  • a further aspect involves an improvement to the above method which comprises recovering RNA from the surfactant-nucleic acid complex formed by the method.
  • This recovery step can include solubilizing the complex with guanidinium salts or with hot formamide.
  • the surfactant can be extracted from its association with the nucleic acid, leaving the RNA insoluble by treating the complex with ethanol and a salt or with a concentrated aqueous solution of lithium chloride.
  • the invention provides a kit for isolating and purifying RNA from a biological sample which contains at least an aqueous surfactant as described herein.
  • the present invention provides a novel surfactant solution useful for extracting RNA from biological samples which comprises a selected quaternary amine salt produced by the reaction of a quaternary amine hydroxide and an acid selected from the group consisting of phosphoric, sulfuric, formic, acetic, propionic, oxalic, malonic, succinic and citric.
  • a selected quaternary amine salt produced by the reaction of a quaternary amine hydroxide and an acid selected from the group consisting of phosphoric, sulfuric, formic, acetic, propionic, oxalic, malonic, succinic and citric.
  • the present invention provides a method for isolating RNA from biological samples which uses a selected novel cationic surfactant comprising a selected quaternary amine, and which method is characterized by significant advantages over methods of the prior art.
  • R3 wherein Rl through R4 are independently selected from an alkyl chain containing from the 1 to 20 carbons, and an aryl group containing from 6 to 26 carbons. Suitable aryl groups are phenyl, lower alkyl-substitute benzyl, and/or halogenated benzyl.
  • Presently preferred anions, i.e., the X" of Formula I, of the quaternary amine surfactants are phosphate, sulfate, formate, acetate, propionate, oxalate, malonate, succinate and citrate.
  • Presently preferred quaternary amine surfactants for use in the RNA isolation method of this invention include the oxalate, malonate and succinate salts of acyltrimethylammonium in which the acyl group is 12, 14 or 16 carbons in length.
  • a presently most preferred surfactant is the oxalate salt of acyltrimethylammonium, wherein the acyl group is 14 carbons in length.
  • Other preferred quaternary amine surfactants for such use include the sulfate, phosphate, formate, acetate and propionate salts of acylbenzyldimethylammonium in which the acyl group is 12, 14, 16 or 18 carbons in length.
  • the surfactants of Formula I which are characterized by the formate, acetate and phosphate salts of hexadecylbenzyldi- methylammonium are also desirable.
  • a novel cationic surfactant useful in the method of the present invention can be obtained as follows: A commercially available surfactant halide in water at about 5 to 30% weight by volume is used as the starting material. Preferably the surfactant halide is at about 15% wt/v in water. A number of commercially available quaternary ammonium halides are available for this purpose from Sigma Chemical Co., including, for example, tetradecyltrimethylammonium bromide.
  • the surfactant halide is converted to the hydroxide by passing through an anion exchange resin prepared in the hydroxide form, such as Dowex 1 (Sigma Chemical) .
  • an anion exchange resin prepared in the hydroxide form, such as Dowex 1 (Sigma Chemical) .
  • Dowex 1 Dowex 1
  • hydroxyl groups on the resin are exchanged for the halide ion.
  • the resulting surfactant hydroxide such as tetradecyltrimethylammonium hydroxide, is assayed by titration.
  • the quaternary ammonium hydroxide is then combined with, and neutralized by, the addition of an acid selected from the group consisting of phosphoric, sulfuric, formic, acetic, propionic, oxalic, malonic, succinic and citric.
  • the resulting quaternary amine surfactant has the formula represented above.
  • the novel surfactants are in an aqueous solution at a concentration of about 0.01 to 0.2 molar at a pH of between 4 and 8.
  • acylbenzyldimethylammonium salts are used as the quaternary amine surfactant in the method of RNA isolation, it is advantageous to add 50mM excess acid.
  • a particularly desirable novel surfactant solution of Formula I. for use in the method described below is also characterized as follows.
  • the surfactant solution is not excessively viscous, i.e., less than 2 cp.
  • the surfactant solution does not crystallize under typical storage conditions, i.e., temperatures of about 0 to 30 C and storage times of about one month.
  • typical storage conditions i.e., temperatures of about 0 to 30 C and storage times of about one month.
  • the surfactant is added to blood in the process of RNA isolation described below, and the mixture is centrifuged, the resulting pellet is of small volume relative to the volume of the blood used in the method, and not dark in color. Additionally, the pellet contains a substantial proportion, that is, greater than about
  • RNA endogenously present in blood or added to the surfactant simultaneously with the blood does not contain substances, such as hemoglobin or its derivatives, in amounts which, after recovery of the RNA as described below, tend to inhibit the action of the reverse transcriptase, DNA polymerase, or other enzymes used in the detection of RNA.
  • a biological sample is mixed with a selected cationic surfactant solution of the invention as described above.
  • Contact of the sample with the surfactant according to this method causes substantially simultaneous lysis of the cells in the sample and precipitation of the RNA in a complex with the surfactant from the lysed cells.
  • the precipitated RNA may be extracted from the complex by either a chaotropic salt and optional phenol extraction, or by a formamide buffer.
  • the complex may be disassociated by solubilizing the surfactant, leaving the RNA insoluble, thereby providing excellent yields of RNA.
  • the surfactant can be solubilized by treating the complex with a concentrated aqueous solution of lithium chloride.
  • the preferred concentration is from about 2 molar to 6 molar lithium chloride.
  • Another treatment which dissociates the complex and solubilizes the surfactant is an ethanolic solution of a salt, such as sodium acetate or lithium chloride.
  • the RNA is further isolated by alcohol precipitation or column chromatography. These methods are discussed in more detail below.
  • RNA includes transfer (t) RNA, ribosomal (r) RNA, and messenger (m) RNA.
  • the method of this invention provides a faster and more convenient method for extracting RNA from biological samples, particularly blood.
  • the method of this invention is rapid; it is possible for the whole procedure to be completed in an hour or less.
  • the RNA obtained by the method, particularly from blood is of an adequate purity such that it is useful for clinical or other uses, such as the use of reverse transcriptase followed by the polymerase chain reaction.
  • the combination can be transported to the laboratory for use in clinical or other analysis without extensive degradation of the RNA.
  • the method of the present invention relies on the use of the novel cationic surfactants identified above.
  • these novel surfactants are unexpectedly efficient in lysing blood and other fluids and tissues containing intact cells and unexpectedly efficient in precipitating RNA. They are also stable on storage, in that they do not precipitate from aqueous solution.
  • a selected biological sample e.g., blood
  • a solution of a selected novel cationic surfactant described herein Generally, in the mixture, 5 to 40 volumes of blood per 100 volumes of surfactant solution are used.
  • the blood and surfactant may be in contact in the mixture for between about 5 minutes to about 24 hours. Presently, a contact time of about 10 minutes is used. No other processing is needed.
  • the quaternary amine surfactant forms an insoluble ionic complex, characterized by hydrophobic binding of the surfactant tails, with the nucleic acids (both DNA and RNA) in the sample.
  • the surfactant/nucleic acid complex is separated from the mixture.
  • the blood-surfactant mixture is centrifuged to precipitate the surfactant/nucleic acid complex. This can conveniently be performed in about 5 to about 30 minutes at about 5000 to about 100,000 g, using about 1 ml samples in an Eppendorf microcentrifuge. When blood is the sample, presently preferred conditions are about 5 minutes at about 16,000g, although any approximately equivalent centrif gation could be used. If the sample is cultured cells, lesser centrifugation times and speeds may be desirable. One of skill in the art can determine the appropriate centrifugation depending on the nature of the biological sample. A suitable alternative to centrifugation is filtration using a filter of about 0.22 micron.
  • the supernatant is removed, and the resulting pellet (or filtrate) , which comprises the surfactant/nucleic acid complex is optionally washed with water.
  • the pellet (or filtrate) is then (1) extracted to solubilize the RNA and dissociate it from its complex with the surfactant or (2) treated to solubilize the surfactant and dissociate it from its complex with the insoluble RNA.
  • a concentrated salt solution is used to extract the RNA from the surfactant/RNA complex.
  • a desirable concentration of salt for this purpose is in excess of 800mM in about one-fifth the volume of the surfactant. It is also advantageous to use salts which inhibit RNases.
  • a particularly suitable salt solution for this purpose includes 4M guanidinium isothiocyanate with lOOmM sodium acetate buffer, at about pH 4.
  • suitable salt solutions could be used in this step, provided that the salt is added in sufficient concentration to dissociate the RNA/surfactant complex.
  • One of skill in the art may select other salts at desired concentrations for this purpose.
  • the separation step may be followed by an alternative step for dissociating the RNA from the nucleotide/surfactant complex.
  • an extracting solvent consisting primarily of formamide, preferably buffered with a suitable salt and acid, may be used to treat the pellet resulting from the separation step described above.
  • a preferred solvent useful for extracting RNA from the surfactant/nucleotide pellet is optimally formamide containing 0-8% w/v sodium acetate or ammonium acetate and 0-1% v/v acetic acid. More preferred is formamide with 4% w/v of the salt and 0.16% v/v acid. The presence of the salt and acid may inhibit RNases.
  • the extraction is carried out at about 25°C to about 100°C for a time period from about 5 to about 30 minutes with occasional vortexing.
  • Presently preferred conditions are 80°C for about 10 minutes with occasional vortexing. Selection of the specific conditions for this step may readily be performed by one of skill in the art.
  • the quality and quantity of the extracted RNA is also enhanced by the optional addition of an RNase inhibitor, such as aurin tricarboxylic acid (0.5-5mM) or diethylpyrocarbonate, to the extracting solvent.
  • RNase inhibitor such as aurin tricarboxylic acid (0.5-5mM) or diethylpyrocarbonate
  • Other inhibitors of RNase may be selected for this purpose by one of ordinary skill in the art.
  • the mixture is optionally centrifuged at rates the same or similar to those indicated above. The supernatant is added to an equal volume of ethanol, and the mixture is cooled to -20°C or below. Thereafter the RNA is centrifuged into a pellet and processed by conventional methods.
  • RNA emerges from the column in the buffer with which the column was equilibrated.
  • the nucleic acid/surfactant complex can be dissociated by methods that leave the RNA insoluble, but which solubilize the surfactant. This can be achieved by washing the pellet with a concentrated aqueous solution of lithium chloride (in which RNA is insoluble) . A preferred concentration is from about 2M to about 6M of lithium chloride. Another method involves washing the pellet with a salt dissolved in ethanol. Preferred salts can include sodium acetate and lithium chloride, although one of skill in the art may select other suitable salts. Where the ethanolic solution contains sodium acetate, a preferred amount of the salt is about 2 to about 10% w/v.
  • a preferred amount of the salt is between about 1 to about 30% w/v.
  • the resulting RNA can be optionally further purified according to this method by phenol/chloroform extraction and precipitated by the addition of ethanol or isopropanol in conventional methods as described by Maniatis et al, and Wallace, both cited above, or by column chromatography. 3. A Kit of the Invention
  • One or more of the above-described surfactant solutions may be readily prepared in a kit for isolating ribonucleic acid from a biological sample.
  • a presently preferred surfactant for such use is acyltrimethylammonim oxalate, with 14 carbons in the acyl group.
  • Additional components of such a kit would include the reagents and containers necessary for the performance of the separating and dissociating steps of this method, i.e., the formamide solvent, the guanidinium isothiocyante solution, the lithium chloride solution and/or ethanolic solution.
  • the reagents for accomplishing the additional purification steps identified above may also be included in such a kit for ready performance of this method.
  • Other conventional components of kits for such isolation methods may also be included in a kit.
  • a surfactant useful in this invention is synthesized as follows. A 15% w/v solution of tetradecyltrimethylammonium bromide (Sigma Chemical
  • surfactants useful in this invention are synthesized as described in Example 1, except that the identities of the quaternary ammonium ion and acid differ as indicated.
  • Each hydroxide was neutralized with an acid selected from the group hydrobromic, hydrochloric, phosphoric, sulfuric, formic, acetic, propionic, oxalic, malonic, succinic and citric, so that the counter ion, X " was bromide, chloride, phosphate, sulfate, formate, acetate, propionate, oxalate, malonate, succinate and citrate in individual surfactants.
  • an acid selected from the group hydrobromic, hydrochloric, phosphoric, sulfuric, formic, acetic, propionic, oxalic, malonic, succinic and citric, so that the counter ion, X " was bromide, chloride, phosphate, sulfate, formate, acetate, propionate, oxalate, malonate, succinate and citrate in individual surfactants.
  • aqueous surfactants were then used in experiments to obtain RNA from blood as described in Example 3.
  • Example 2 Surfactant solutions synthesized as described in Example 2 (1 ml) were placed in Eppendorf microcentrifuge tubes. Two hundred microliters of blood anticoagulated with 1/10 vol 3.2% sodium citrate was added with immediate mixing. The mixture was centrifuged at 16,000 g for 5 minutes, and the supernatant was removed by aspiration.
  • the resulting pellet was examined visually and graded.
  • the results are provided in the following table, where 12, 14, or 16-TMA indicates acyltrimethylammonium with acyl of 12, 14, or 16 carbons in length, and 12, 14, 16, or 18-BA indicates acylbenzyldimethylammonium with acyl of 12, 14, 16, or 18 carbons in length.
  • the counter ions of the surfactants are indicated along the left side of the table.
  • Samples of blood (100 microliters and 400 microliters) were added to 800 microliters 14-TMA oxalate prepared as in Example 1 in an Eppendorf tube with immediate mixing. The mixture was incubated at room temperature for 0, 15 minutes, 30 minutes or 1 hour before centrifugation (5 minutes at 16,000 g) . The supernatant was aspirated and the pellet was washed briefly with RNase-free water.
  • the pellet was then extracted with an extracting buffer, produced by mixing 4 grams sodium acetate and 0.16 mL acetic acid with 100 ml formamide, by heating at 80°C for 10 minutes with occasional vortexing.
  • the mixture was centrifuged (16,000 g, 5 minutes), and the supernatant was added to 400 microliters ethanol and cooled to -80°C for 10 minutes.
  • the precipitated RNA was harvested by centrifugation (16,000 g, 5 minutes), dissolved in a formaldehyde sample buffer and electrophoresed in agarose by a conventional technique. Examination of the gel under ultraviolet light after staining with ethidium bromide showed the presence of rRNA and other RNA in the lanes loaded with the 100 microliter samples of blood.
  • the lanes loaded with 400 microliters blood revealed RNA that was partially degraded. There was no difference between the lanes containing samples incubated with the surfactant for 0, 15 minutes, 30 minutes, or 1 hour.
  • RNA was isolated from blood using the surfactants listed in Example 3 with pellet scores of 0 or 1. Yields with the benzalkonium surfactants were generally lower than with the alkyltrimethylammonium surfactants. The reason for this is not clear.
  • Example 6 RNA Isolation and Extraction from a Cell Suspension
  • HL-60 ATCC CCL 240
  • K562 ATCC CCL 243
  • human leukemia cells in 100 microliters were added to 1 ml 100 mM 14-TMA oxalate and centrifuged (16,000 g, 5 minutes). The pellet was extracted and analyzed as described in Example 5. Characteristic bands of rRNA were seen on the gel, as well as other RNA species. This demonstrates that TMA oxalate can be used to isolate RNA from cultured cells.
  • RNA samples of blood were added to 1.0 ml 14-TMA oxalate with or without 10 or 100 mM dithiothreitol, and after centrifugation the pellet was extracted as described in Example 6 with formamide containing 0, 10 or 100 mM dithiothreitol.
  • the RNA was precipitated with ethanol and examined by agarose gel electrophoresis. The best yield of undegraded RNA was obtained when 100 mM dithiothreitol was added to both surfactant and formamide extracting buffer. Dithiothreitol may increase yields by inhibiting RNase.
  • Example 9 Purification of Extracted RNA Using Column Chromatography
  • Example 8 An experiment similar to Example 8 was performed in which blood and radioactive RNA was added simultaneously to the surfactant, except that the formamide extract of the RNA was chromatographed on one of several size exclusion columns pre-equilibrated with an aqueous buffer (Sephadex G50TM or Trisacryl GF-05TM) and eluted by gravity feed, centrifugation or by air pressure. The radioactive fraction emerging from the column eluted with air pressure was analyzed by agarose gel electrophoresis which revealed bands of undegraded rRNA. This experiment reveals that RNA can be recovered from formamide by column chromatography.
  • an aqueous buffer Sephadex G50TM or Trisacryl GF-05TM
  • Samples of blood (50-400 microliters) were added to 1 ml 14-TMA oxalate solution, and centrifuged at 16,000 g for 5 minutes). The resulting pellets were extracted with 100 microliters of an aqueous solution containing 4 M guanidinium isothiocyanate and 200 mM sodium acetate buffer, pH 4 by incubation at room temperature for 10 minutes with occasional vortexing. An equal volume of a 1:1 mixture of water-equilibrated phenol and chloroform was then added, and emulsified by vortexing. The phases were separated by brief centrifugation (16,000 g, 2 minutes), and the upper aqueous layer was removed, and added to an equal volume of isopropanol.
  • RNA was extracted from the surfactant nucleic acid pellet by high salt concentrations. Guanidinium isothiocyanate is known to inhibit RNase, which action may facilitate the recovery of RNA.
  • Chronic myelogenous leukemia cells express an oncogene (bcr/abl ) which is a hybrid of two genes juxtaposed by a reciprocal translocation between two chromosomes. This oncogene is not expressed in normal cells, but is expressed in the immortal leukemic cell line K562 [ATCC CCL 243].
  • bcr/abl oncogene
  • B Maloney murine leukemia virus reverse transcriptase
  • RNasin 5 mM dithiothreitol
  • 20 pmol primer B S'-TCAGACCCTGAGGCTCAAAGTC
  • reaction was stopped by heating to 95°C. 80 microliters of PCR buffer and 20 pmol of primer A (5'-GAAGCTTCTCCCTGGCATCCGT-3 » ) [SEQ ID NO: 2] was added. The mixture was overlaid with 100 microliters mineral oil and programmed to cycle. All procedures were modified from those published by using the "hot-start" technique.
  • the Thermal Cycler Perkin Elmer-Cetus, Emeryville, CA was programmed as follows: denature at 95°C for 30 seconds, anneal at 55°C for 30 seconds and extend at 72°C for 1 minute when primers A and B were used. PCR products were analyzed on 1% agarose gels with ethidium bromide.
  • Example 11 An experiment similar to Example 11 was performed, except that the guanidinium method of Example 10 was used to isolate the RNA. An amplified product of the appropriate size was seen when RNA from blood samples contained 30 or more K562 cells. This experiment shows that extracting the surfactant nucleotide complex with guanidinium isothiocyanate as described yields RNA which is suitable for amplification without further purification. As described, this method would appear to be capable of detecting less than one leuke ic cell per microliter of blood of patients with chronic myelogenous leukemia having the Philadelphia chromosome, and this illustrates the great sensitivity of this method.
  • HL60 or K562 cells (10 5 - 10 7 cells) were added to 1 ml 14-TMA oxalate, and the mixture was centrifuged as described in Example 6. The pellet was mixed with 2M aqueous lithium chloride, and centrifuged again, discarding the supernatant. The pellet containing the RNA was washed with 70% ethanol, and dissolved in an aqueous buffer. Examination by UV spectroscopy and agarose gel electrophoresis revealed an excellent yield of undegraded RNA.
  • RNA was isolated from cultured cells as described in Example 13 except that ethanol containing 4% sodium acetate was substituted for the lithium chloride solution. Again, an excellent yield of largely undegraded RNA was obtained.
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:2: GAAGCTTCTC CCTGGCATCC GT 22

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Abstract

La présente invention concerne un nouveau procédé d'isolation de l'ARN à partir de prélèvements biologiques, notamment de sang, à l'aide d'agents tensioactifs aminés quaternaires. L'ARN est isolé de manière rapide et en une quantité et une qualité suffisantes pour permettre l'analyse, en faisant appel à des procédés comprenant la transcriptase réverse et la PCR.
PCT/US1994/000680 1993-02-01 1994-01-12 Agents tensioactifs amines quarternaires et procedes d'utilisation de ceux-ci WO1994018156A1 (fr)

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JP51806594A JP3615545B2 (ja) 1993-02-01 1994-01-12 第四級アンモニウム塩界面活性剤及びそのｒｎａの単離剤
AU62305/94A AU6230594A (en) 1993-02-01 1994-01-12 Quartenary amine surfactants and methods of using same in isolation of rna

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US08/013,419 US5300635A (en) 1993-02-01 1993-02-01 Quaternary amine surfactants and methods of using same in isolation of nucleic acids
US11372793A 1993-08-27 1993-08-27
US08/113,727 1993-08-27
US08/013,419 1993-08-27

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Cited By (13)

* Cited by examiner, † Cited by third party
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WO1999029840A1 (fr) * 1997-12-09 1999-06-17 The Perkin-Elmer Corporation Methodes d'isolement de l'acide nucleique
EP1031626A1 (fr) * 1999-02-23 2000-08-30 QIAGEN GmbH Procédé pour la stabilisation et/ou l'isolement d'acides nucléiques
EP1329506A1 (fr) * 2002-01-18 2003-07-23 Cypro S.A. Procédé de quantification du niveau in vivo d'ARN
US6602718B1 (en) 2000-11-08 2003-08-05 Becton, Dickinson And Company Method and device for collecting and stabilizing a biological sample
WO2002056030A3 (fr) * 2000-11-08 2003-08-28 Becton Dickinson And Company Methode et dispositif de prelevement et de stabilisation d'un echantillon biologique
WO2003072816A2 (fr) * 2002-02-26 2003-09-04 Qiagen Gmbh Procede de modification de la concentration de transcription dans des echantillons biologiques contenant de l'acide ribonucleique
WO2005123960A1 (fr) * 2004-06-11 2005-12-29 Ambion, Inc. Derives biologiques bruts adaptes pour la detection des acides nucleiques
WO2006103094A2 (fr) 2005-04-01 2006-10-05 Qiagen Gmbh Procede pour traiter un echantillon contenant des biomolecules
WO2010108989A1 (fr) 2009-03-27 2010-09-30 Université Libre de Bruxelles Nouveau marqueur pour le diagnostic d'une sclérose en plaques active
WO2011051402A1 (fr) 2009-11-02 2011-05-05 Universite Libre De Bruxelles Nouveaux biomarqueurs pour déterminer un état d'allergie
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US5728822A (en) 1998-03-17

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